Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
  • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/154—Methylation markers
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/158—Expression markers

Definitions

• the present invention is in the field of molecular diagnostics.
• the invention provides diagnostic markers, methods, systems, and kits for detecting a predisposition to, or the presence of colorectal cancer (CRC), such as detecting a change in the expression status or methylation status, or a combination thereof of any one or more of a number of genes.
• CRC colorectal cancer
• the markers disclosed herein provide a higher sensitivity and/or specificity in comparison to conventional markers.
• Colorectal cancer also known as bowel cancer and colon cancer, is the development of cancer from the colon or rectum (parts of the large intestine).
• a cancer is the abnormal growth of cells that have the ability to invade or spread to other parts of the body. Signs and symptoms may include blood in the stool, a change in bowel movements, weight loss, and general fatigue.
• colorectal cancers are due to old age and lifestyle factors with only a small number of cases due to underlying genetic disorders. Some other risk factors include diet, obesity, smoking, and lack of physical activity. Dietary factors that increase the risk include the consumption of red and processed meat as well as alcohol. Another risk factor is inflammatory bowel disease, which includes Crohn's disease and ulcerative colitis. Some of the inherited genetic disorders that can cause colorectal cancer include familial adenomatous polyposis and hereditary non-polyposis colon cancer; however, these represent less than 5% of cases. Colorectal cancer typically starts as a benign tumor, often in the form of a polyp, which over time becomes cancerous.
• the current gold standard diagnostic test for colorectal cancer is colonoscopy, an invasive procedure during which a trained gastroenterologist checks the rectum and colon for polyps and other types of abnormal tissues and removes them if necessary. Tissue biopsies may also be collected that can then be inspected by a pathologist to determine whether they are pre-cancerous or cancerous. Much effort has been exerted into the development of minimally or non-invasive diagnostic tests that can precede or even replace colonoscopies, reducing the number of unnecessary invasive procedures and potentially increasing the participation rates in population-based screening programs.
• DNA methylation an epigenetic mechanism that controls gene expression through the chemical binding of methyl, a small molecule, to specific genomic locations.
• Hypomethylation in general, arises earlier and is linked to chromosomal instability and loss of imprinting, whereas hypermethylation is typically associated with promoters (or enhancers) and can result in (tumor suppressor) gene silencing, which also provides a target for epigenetic therapy.
• hypermethylation in tumor suppressor genes and hypomethylation of oncogenes.
• the presence of DNA methylation in the promoter region of a gene most often prevents the transcription of this gene.
• DNA methylation is a vital biological process involved in many developmental processes, while aberrant DNA methylation patterns are found in virtually every type of human cancer.
• Cologuard® test achieved a sensitivity of 92.3% and specificity of 89.9% for detecting colorectal cancer, compared to 73.8% and 96.4% for the fecal immunochemical test (FIT; Imperiale et ak, 2014, clinical trial NCT01397747). While Cologuard® offers higher sensitivity than FIT, it also results in more false positives [1].
• ProColon® test achieved a sensitivity of 73.3% and a specificity of 81.5%, compared to 68.0% and 97.4% for FIT (Johnson et ak, 2014). In this case, the specificity was lower for the DNA-methylation based test than for FIT.
• the advantage of the Epi ProColon® test is that it is blood-based and could therefore increase screening participation. A study in Germany showed that of the people that refuse to undergo a colonoscopy, 97% accepted a non-invasive alternative (such as a stool or blood-based test) and that of these 97%, 83% preferred a blood test (Adler et ak, 2014).
• Noninvasive methods may also help in detecting cancer at very early stages, even before they become malignant, if one knows the correct markers for evaluation. Pre-cancer that goes unchecked may ultimately become cancerous. Methylation-based tests may be helpful in identifying increased risk of cancer, which should serve as a reminder to stay current with medical visits and screening tests and communicate concerns or changes to the doctor. Improved sensitivity of such tests and methods can for instance be a huge contributor in identifying dysplastic adenoma and other high-risk adenoma’s that will likely evolve to malignant CRC.
• the clinician has to take the next step in the clinical management, which may include determining the prognosis for this patient or estimating the likely progression of the cancer and the aggressiveness that it may exhibit (recurrence likelihood, progression and/or chance for metastasis despite adjuvant therapy). The clinician then may tailor the treatment and therapy to this likely evolution of the disease.
• the current practice for prognosis assessment is based on radiological (CT, MRI) and pathological (TNM, lymphovascular, perineural, and venous invasion) criteria.
• CT radiological
• TNM pathological
• markers has the robustness to be translated in clinical practice [12]
• certain biomarkers can be used to detect minimal residual disease (MRD) and determine whether treatment and/or therapy is working, or if cancer has recurred, allowing the clinician to potentially adjust the patient management.
• MRD minimal residual disease
• the final step in the clinical management of a patient with cancer is the determination of the most suitable therapeutic regimen. It is well known that patients with cancer exhibit different responses to certain therapies and some patients can benefit the most from the use of a biologic factor. Thus, it is of importance to be able to predict the most likely response to a given therapy. For this reason, predictive biomarkers are being evaluated. But, also here, more accurate biomarker panels are needed for use in clinical settings.
• non-invasive methods and their biomarkers for evaluation may be helpful in early detection, diagnosis, therapy choices, patient stratification for therapy, treatment monitoring, detection of minimal residual disease, and recurrence monitoring.
• One object underlying the present invention is therefore to find a marker or set of markers that improve the sensitivity and/or specificity of tests in the field of CRC.
• the marker and set of markers find their utility in screening, monitoring, stratification, and therapeutic set ups and find their applications in kits suitable for such utility.
• the disclosed subject matter relates to methods, systems, and kits for detecting alterations in expression status of one or more genes in a sample containing or suspected of containing colorectal tumor or colorectal cancer nucleic acid taken from a human subject.
• the methods may comprise of, and the systems and kits may be used for, performing an assay on the sample and detecting expression status of at least one gene in the sample that differs from a reference expression status, wherein the at least one gene is selected from the genes listed in Table 1.
• the disclosed subject matter concerns methods, systems, and kits for detecting a predisposition to, or the presence of colorectal cancer in a sample containing or suspected of containing colorectal tumor or colorectal cancer nucleic acid taken from a human subject comprising detecting alterations in expression status in said sample, comprising: performing an assay on the sample and detecting the expression status of at least one gene that differs from a reference expression status, wherein the at least one gene is selected from the genes listed in Table 1, and, wherein the detection of an altered expression is indicative of a predisposition to, or the presence of colorectal cancer.
• the disclosed subject matter also concerns methods, systems, and kits for determining a prognosis of colorectal cancer and/or determining a predisposition to colorectal cancer in a sample containing or suspected of containing colorectal tumor or colorectal cancer nucleic acid taken from a human subject comprising detecting alterations in expression status in said sample, comprising: performing an assay on the sample and detecting the expression status of at least one gene that differs from a reference expression status, wherein the at least one gene is selected from the genes listed in Table 1, and, wherein the detection of an altered expression is indicative for cancer development, likely evolution of the disease, or predisposition to colorectal cancer.
• the disclosed methods, systems, and kits may be performed in order to characterize a stage of colorectal cancer development.
• the disclosed subject matter also provides methods, systems, and kits for detecting alterations in methylation status in a sample containing or suspected of containing colorectal tumor or colorectal cancer nucleic acid taken from a human subject, the method comprising: performing an assay on the sample and detecting a methylation status of at least one gene that differs from a reference methylation status, wherein the at least one gene is selected from the genes listed in Table 1.
• the disclosed subject matter also relates to methods, systems, and kits for detecting a predisposition to, or the presence of colorectal cancer in a sample containing or suspected of containing colorectal tumor or colorectal cancer nucleic acid taken from a human subject comprising detecting alterations in methylation status in said sample, comprising: performing an assay on the sample and detecting the methylation status of at least one gene that differs from the reference methylation status, wherein the at least one gene is selected from the genes listed in Table 1, and, wherein the detection of an altered in methylation status is indicative of a predisposition to, or the presence of colorectal cancer.
• the disclosed subject matter also concerns a method of prognosis to colorectal cancer or predisposition to cancer in a sample containing colorectal tumor or colorectal cancer nucleic acid taken from a human subject comprising detecting alterations in methylation status in said sample, comprising: performing an assay on the sample and detecting alterations in methylation status of at least one gene that differs from the reference methylation status, wherein the at least one gene is selected from the genes listed in Table 1, and, wherein the detection of alterations in methylation status is indicative for cancer development, likely evolution of the disease, whether pre- or post-treatment, or predisposition to colorectal cancer.
• kits for detecting alterations in expression status in a sample containing colorectal tumor or colorectal cancer nucleic acid comprising: tools to specifically detect the methylation status or promotor methylation status of at least one of the genes listed in Table 1.
• the gene or genes for use in the disclosed methods, systems, and kits may be chosen from: TRBJ2-2, TRBJ2-4, SLC18A3, RIPPLY2, GALR1, FAM19A4, SYT9, KCNA1, TMEM196, VSX1, MMD2, GPM6A, SNAP91, PRKG2, SLC27A6, UNC5D, NOS1, TRHDE, STOX2, C12orf56, GALNT13, TMEM130, GPR27, CRTAC1, NRG3, PTPRT, NLGN4X, SLITRK5, RIMS2, COL25A1, CNNM1, EIF4E3, VIPR2, KCNIP4, SLC35F3, ZNF542, LRRC7, STK32B, TRBJ2-2P, FAM184B, PREX2, CACNA1A, GLB1L3, PCDH7, PDE6B, COL23A1, NCAM2, FBLIM1, KRBAl, KY, AMPH, ZNF132, ATP8
• Figure 1 Example distribution of the AUC values of all 1540 three-marker panels, including the AUC cutoff, for the comparison of normal and CRC samples.
• CRC Colorectal cancer
• the purpose of the invention is to provide accurate biomarkers for diagnostic, prognostic and predictive purposes that either alone or as part of a panel would help improve the patient's clinical management.
• biomarkers from the list in three solid tissue sample types: i) normal tissue, ii) adenoma tissue (adenomas are non-malignant lesions that can develop into colorectal cancer), and ii) colorectal cancer tumor tissue.
• normal tissue i) normal tissue
• adenoma tissue adenomas are non-malignant lesions that can develop into colorectal cancer
• colorectal cancer tumor tissue ii)
• the same biomarkers have also been tested in stool samples from people with the same three conditions (healthy, adenoma, colorectal cancer).
• the disclosed subject matter thus generally relates to novel biomarkers, biomarker panels, methods exploiting the markers and panels, and systems and kits for performing said methods for analyzing colorectal tumor or colorectal cancer samples taken from a human subject.
• the disclosed subject matter relates to methods, systems, and kits for detecting alterations in expression status in a sample containing or suspected of containing colorectal tumor or colorectal cancer nucleic acid taken from a human subject.
• the methods may comprise and the systems and kits may be utilized for performing an assay on the sample and detecting the expression status of at least one gene that differs from a reference expression status; wherein the at least one gene is selected from the genes listed in Table 1.
• the disclosed subject matter also relates to methods, systems, and kits for determining a predisposition to, or tire presence of colorectal cancer in a sample containing or suspected of containing colorectal tumor or colorectal cancer nucleic acid taken from a human subject comprising detecting alterations in expression status in said sample, comprising: performing an assay on the sample and detecting the expression status of at least one gene that differs from a reference expression status, wherein the at least one gene is selected from the genes listed in Table 1, and, wherein the detection of an altered expression is indicative of a predisposition to, or tire presence of colorectal cancer.
• the reference expression status will be a predetermined reference expression status.
• the reference expression status, predetermined expression status, or normal expression value is usually referred to as “cut-off value”, “reference value”, “reference level”, or “predetermined reference value”.
• the disclosed subject matter relates to methods, systems, and kits for determining whether a subject has a predisposition to, or the presence of colorectal cancer (CRC) comprising the steps of: determining the expression status from one or more genes listed in Table 1 in a sample from the subject and comparing said expression status of each of said genes with a predetermined reference status of each of said genes, wherein it is concluded that the subject has a predisposition to, or the presence of CRC if at least one of said genes have an altered expression status relative to the predetermined reference status of at least one of said genes.
• CRC colorectal cancer
• the subject has a predisposition to, or the presence of CRC if at least two, three, or more of said genes has an altered expression status relative to the predetermined reference status of at least two, three, or more of said genes.
• a skilled person may be well aware of ways of obtaining such a reference expression status or predetermined reference expression status. It may for instance be the value obtained using the same probes and methods as described herein when applied to a normal individual or a panel of normal individuals. It may also be an arbitrary chosen value or it may be determined by trial and error.
• predetermined reference expression status means the expression status of a particular gene when measured as described above in a sample from an individual not suffering from CRC.
• Suitable normal subjects may include a subject that is negative for any colon abnormality in colonoscopy.
• Suitable subjects may include human subjects.
• the reference value or predetermined reference status may also be determined empirically by the skilled person.
• the skilled person is well aware of the ins and outs of determining a reference status for measuring and determining the expression status of a gene.
• the disclosed subject matter also concerns methods, systems, and kits for determining a prognosis of colorectal cancer and/or determining a predisposition to colorectal cancer in a sample containing or suspected of containing colorectal tumor or colorectal cancer nucleic acid taken from a human subject comprising detecting alterations in methylation status in said sample, comprising: performing an assay on the sample and detecting alterations in methylation status of at least one gene that differs from a reference methylation status, wherein the at least one gene is selected from the genes listed in Table 1, and, wherein the detection of an alteration in methylation status is indicative for cancer development or predisposition to colorectal cancer.
• Such method of prognosis of colorectal cancer can be applied pre- or post treatment. Testing can be performed diagnostically or in conjunction with a therapeutic regimen. Testing can be used to determine what therapeutic or preventive regimen to employ on a patient and be used to monitor efficacy of a therapeutic regimen or treatment/ surgery .
• the disclosed subject matter also provides a method for predicting the likelihood of successful treatment of a cancer as defined herein, such as CRC, comprising measurement of alterations in expression status and/or methylation status of at least one gene that differ from the reference expression and/or methylation status, wherein the at least one gene is selected from the genes listed in Table 1, wherein an altered expression status and/or methylation status indicates the efficacy of treatment/likelihood of resistance to treatment.
• the method for predicting the likelihood of successful treatment of a cancer as defined herein, such as CRC comprises measurement of alterations in expression status and/or methylation status of at least one gene that differ from the reference expression and/or methylation status, wherein the at least one gene is selected from the genes listed in Table 1, wherein an un-altered expression status and/or methylation status indicates the efficacy of treatment/likelihood of resistance to treatment.
• Table 1 List of highly sensitive and specific biomarkers with alterations in their expression statuses, applicable for tests in the field of CRC.
• Nomenclature Committee which is responsible for approving unique and unambiguous symbols and names for human genes.
• a combination of markers from the established list of highly sensitive and specific biomarkers may be utilized.
• the disclosed methods may also be performed using the expression status of at least two, three, four, five, six, seven, eighth, nine, ten genes, or more genes from the genes listed in Table 1.
• a panel of genes is used to obtain a better specificity and/or sensitivity of the method.
• the determination of the expression status of a panel of genes may be part of a multiplex set up in which all genes of the panel are simultaneously assessed for their expression. Whereas multiplexing undoubtedly has certain advantages, looking at one gene at a time may be necessary in order to solve certain technical challenges.
• one or more of the genes are separately assessed for their expression status. In such case, a combination of the separate read-out results is used as basis for a colorectal cancer indication.
• the at least one, two, three, four, five, six, seven, eighth, nine, ten, or more gene(s) is (are) preferably chosen from these 29 genes.
• Such a population comprises a number of false positives that may then be submitted to an assay according to the disclosed methods. This will then detect the false positives and identify them correctly as CRC negatives. It may also be possible to combine FIT with methylation markers and balance its non-specificity in an algorithm.
• the panel comprises any one, two or three of the genes ADHFE1, AMPH, FAM184B, FAM19A4, FBLIM1, GALNT13, GDNF, GFRA1, HAND2, ITGA4, KCNIP4, LONRF2, LRRC7, MARCHF11, NALCN, NDRG4, NLGN4X, PCDH7, SLC27A6, SLC35F3, SLC6A2, SNAP91, SORCS1, SYT9, TM6SF1, TMEM130, TRHDE, UNC5C, and VIPR2.
• the methods may comprise determining the expression status of a panel of genes comprising the at least two genes, up to three genes, up to four genes, up to five genes, ... up to ten genes.
• the invention relates to methods, systems, and kits for detecting a colorectal cancer as described herein wherein the indication of colorectal cancer is based on the detection of an altered expression status of said gene or said genes from Table 1. More particularly, detection of an altered expression status of at least one or more of the genes ADHFE1, AMPH, FAM184B, FAM19A4, FBLIM1, GALNT13, GDNF, GFRA1, HAND2, ITGA4, KCNIP4, LONRF2, LRRC7, MARCHF11, NALCN, NDRG4, NLGN4X, PCDH7, SLC27A6, SLC35F3, SLC6A2, SNAP91, SORCS1, SYT9, TM6SF1, TMEM130, TRHDE, UNC5C, and VIPR2, may lead to such indication.
• colorectal cancer is indicated if at least two of said genes have an altered expression status relative to the respective reference expression status of said at least two genes.
• Suitable combinations of genes may include ADHFE1 and ITGA4, or, ADHFE1 and LONRF2, or, ITGA4 and LONRF2.
• colorectal cancer is indicated if at least three of said genes have an altered expression status relative to the respective reference expression status of said at least three genes.
• the disclosed subject matter also relates to a method as described herein wherein it is concluded that the subject has CRC if all three of genes ITGA4, ADHFE1, and LONRF2 have an altered expression, for instance a lower expression status relative to the respective reference expression status of said three genes.
• the expression status of the genes may be determined in a number of ways, all well known to the skilled person. It may be done by determining the RNA level expressed by these genes or even the level of expression of the proteins encoded by these genes. For gene expression analysis there are a number of routine techniques available such PCR-based techniques, mRNA northern blotting, nuclease protection assays, in situ hybridization, microarrays and many more. Protein expression may be detected by western blotting or immunohistochemistry. Within the category of PCR-based approaches, both end-point PCR and real-time PCR have been used, and the most common used PCR-based approach is quantitative RT-PCR.
• the expression status of the one or more genes under investigation is determined by measuring the DNA methylation status of the gene or DNA methylation status of their promoters relative to a normal status, cut-off level or any other suitable reference level or predetermined reference level. Whenever the term “methylation” is used herein, it refers to DNA methylation. In one embodiment, at least one gene of which the DNA methylation status or the DNA methylation status of its promoter is determined is chosen from the genes listed in Table 1.
• At least one gene of which the DNA methylation status or the DNA methylation status of its promoter is determined is chosen from the subset of genes ADHFE1, AMPH, FAM184B, FAM19A4, FBLIM1, GALNT13, GDNF, GFRA1, HAND2, ITGA4, KCNIP4, LONRF2, LRRC7, MARCHF11, NALCN, NDRG4, NLGN4X, PCDH7, SLC27A6, SLC35F3, SLC6A2, SNAP91, SORCS1, SYT9, TM6SF1, TMEM130, TRHDE, UNC5C, and 17/7/2.
• the methylation status is determined in suitable CpG islands, or other CpG-rich regions, which are often found in the promoter, and enhancer, region of the gene(s).
• the term “methylation”, “methylation state”, “methylation level”, or “methylation status” refers to the presence or absence of 5-methylcytosine ("5-mCyt") at one or a plurality of CpG dinucleotides within a DNA sequence. CpG dinucleotides are typically concentrated in the promoter regions and exons of human genes.
• a reference methylation value or predetermined reference methylation value is for instance a value of 0.2 or more, such as 0.5 or more on the Infmium platform output.
• the value may also be determined with a suitable probe set, such as for instance probes comprising a sequence according to SEQ ID NO: 9, SEQ ID NO: 12, or SEQ ID NO: 15 for ITGA4, ADHFE1, and LONRF2 respectively.
• Probes and their sequences suitable for each of their corresponding genes ADHFE1, AMPH, FAM184B, FAM19A4, FBLIM1, GALNT13, GDNF, GFRA1, HAND2, ITGA4, KCNIP4, FONRF2, FRRC7, MARCHF11, NAFCN, NDRG4, NFGN4X, PCDH7, SFC27A6, SFC35F3, SFC6A2, SNAP91, SORCS1, SYT9, TM6SF1, TMEM130, TRHDE, UNC5C, and VIPR2 are listed in Table 10.
• the predetermined reference methylation level refers to the promoter (or enhancer) methylation level (PMF) of the gene under the control of that promoter (or enhancer) in a sample from a subject not having CRC.
• the reference value may also be determined empirically by the skilled person. The skilled person is well aware of the ins and outs of determining reference values for measuring and determining the level of their promoter methylation.
• promoter methylation level of the genes ITGA4, ADHFE1, and LONRF2 was determined (examples 1 and 2).
• the promoter methylation level (PML) is inversely correlated with the expression level of the gene, i.e. the higher the PML, the lower the expression level of the gene.
• Table 2 shows a comparison of the sensitivity and specificity of the disclosed methods versus the commercially available Cologuard assay.
• Table 4 Results from Table 3 scored as positive (+) or negative (-) relative to the predetermined reference value or cut-off value. Samples were scored positive if the PML of at least one/two/three of the three markers was above the cut-off value.
• Table 6 Results from Table 5 scored as positive (+) or negative (-) relative to the cut-off value. All samples were negative for all 3 markers (sum).
• the methylation level of the promoter regions of genes ADHFE1, ITGA4, and LONRF2 may be determined using different techniques at different CpG methylation sites within one or more promoter (or enhancer) regions.
• the promoter regions of the genes ITGA4, ADHFE1, and LONRF2 are herein provided as SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5 respectively. Suitable regions within these promoter regions for Q-PCR analysis may include but are not limited to the regions defined by SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6.
• SEQ ID NO: 47 SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO:
• SEQ ID NO: 34 SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38,
• SEQ ID NO: 39 SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43,
• ADHFE1, ITGA4, UNC5C, GFRA1, SORCS1, SNAP91, SLC27A6, HAND2, GDNF), 19 top ranked CRC methylation biomarkers from Table 1 were evaluated in stool samples from 49 patients that were cancer-free based on the colonoscopy results, 25 patients with adenoma, 17 patients with high-grade adenoma, and 42 patients with confirmed colorectal cancer (Example 2).
• SLC27A6, ADHFE1, and LONRF2 were included for reference performance, totaling 22 genes.
• a ranking of the genes is determined by combining both the delta Ct method rank and the copy number rank: SLC35F3, TM6SF1, FBLIM1, LONRF2, SLC27A6, SLC6A2, KCNIP4, FAM19A4, VIPR2, TMEM130, MARCHF11, ADHFE1, AMPH, TRHDE, LRRC7, GALNT13, NALCN, SYT9, PCDH7, NLGN4X, FAM184B, and NDRG4.
• the disclosed methods may be performed on any sample taken from a subject; however, it is advantageous to use stool samples because they are easily obtainable. Tissue samples, for instance from a colon biopsy may also be advantageously employed because they contain a relatively large amount of tumor DNA. Other types of samples may be used in the disclosed methods. Non-limiting examples include human or animal fresh tissue samples, frozen tissue samples, tissue samples embedded in FFPE (formalin-fixed, paraffin-embedded tissue), whole blood, blood plasma, blood serum, urine, stool, lymph fluid, and rectal swabs. The samples may be collected using any suitable methods known in the art.
• FFPE formalin-fixed, paraffin-embedded tissue
• the sample may be a tissue sample, swab specimen, body fluid, body fluid precipitate or lavage specimen.
• the sample under investigation contains nucleic acid from a colorectal tumor or colorectal cancer to be analyzed according to the disclosed methods.
• the disclosed subject matter also provides systems and kits for performing the disclosed methods.
• the disclosed subject matter provides systems and kits for detecting alterations in expression status in a nucleic acid containing colorectal tumor or colorectal cancer sample, the systems and kits comprising: components, reagents, and/or tools to specifically detect methylation levels or promotor methylation levels of at least one of the genes listed in Table 1.
• the disclosed components, reagents, and/or tools may be sequence-specific and/or may be utilized for detecting expression of at least one gene listed in Table 1 in a sequence specific-manner.
• the disclosed components, reagents, and/or tools may be utilized for detecting promotor methylation of at least one gene listed in Table 1.
• the disclosed components, reagents, and/or tools may include, but are not limited to primers, probes, and other reagents such as a proofreading polymerase.
• primers and probes suitable for the detection of colorectal cancer according to the disclosed methods, systems, and kits are disclosed in the description and comprise: a probe according to SEQ ID NO: 9 and primers according to SEQ ID NO: 7 and SEQ ID NO: 8 for measuring the methylation level of the promoter of gene ITGA4 a probe according to SEQ ID NO: 12 and primers according to SEQ ID NO: 10 and SEQ ID NO: 11 for measuring the methylation level of the promoter of gene ADHFE1 ; and a probe according to SEQ ID NO: 15 and primers according to SEQ ID NO: 13 and SEQ ID NO: 14 for measuring the methylation level of the promoter of gene LONRF2; a probe according to SEQ ID NO: 113 and primers according to SEQ ID NO: 69 and SEQ ID NO: 91 for measuring the methylation level of the promoter of gene TRHDE; a probe according to SEQ ID NO: 114 and primers according to SEQ ID NO: 70 and SEQ ID
• the disclosed systems and kits may comprise one or more components, reagents, and/or tools for detecting the expression of at least one gene listed in Table 1.
• the disclosed systems and kits comprise one or more components, reagents, and/or tools for detecting the methylation status of at least one gene listed in Table 1, and optionally for detecting the methylation status of multiple genes listed in Table 1.
• Suitable components, reagents, and/or tools for detecting the methylation status of at least one gene listed in Table 1 may include, but are not limited to, components, reagents, and/or tools for performing one or more of the following processes: PCR, Q- PCR, sequencing, hybridization arrays, restriction enzyme approaches (.e.g, methylation- specific PCR (MSP), quantitative methylation-specific PCR (Q-MSP), Illumina sequencing technology, Oxford Nanopore sequencing technology, Ion TorrentTM sequencing technology, Dreaming technology, and High Resolution Melting (HRM) technology).
• MSP methylation- specific PCR
• Q-MSP quantitative methylation-specific PCR
• Illumina sequencing technology Oxford Nanopore sequencing technology
• Ion TorrentTM sequencing technology Ion TorrentTM sequencing technology
• Dreaming technology High Resolution Melting
• Suitable components, reagents, and/or tools may include, but are not limited to reagents that convert unmethylated cytosines to uracil but that do not convert methylated cytosines to uracil (e.g., bisulfite reagents); sequence-specific primers for performing PCR of at least one gene listed in Table 1, either before or after the gene is treated with a reagent that converts unmethylated cytosines to uracil but that does not convert methylated cytosines to uracil (e.g., bisulfite reagents); sequence-specific probes for detecting at least one gene listed in Table 1 and/or a PCR product thereof (e.g., an amplicon thereof) either before or after the gene or the PCR product thereof is treated with a reagent that converts unmethylated cytosines to uracil but that does not convert methylated cytosines to uracil; primers for sequencing a gene listed in Table 1 or a
• the disclosed systems may be designed for detecting the expression status or the methylation status of at least one gene listed in Table 1 in a biological sample from a subject, and optionally designed for detecting the expression status or the methylation status of multiple genes listed in Table 1 in a biological sample from a subject.
• the disclosed systems may comprise at least one hardware processor that is programmed to perform at least one step for detecting the expression status or the methylation status of at least one gene listed in Table 1 in a biological sample from a subject and/or a hardware processor that is programmed to actuate at least one mechanical component of the system to perform one or more steps for detecting the expression status or the methylation status of multiple genes listed in Table 1 in a biological sample from a subject.
• the hardware processor may be configured to perform and/or to actuate one or more mechanical components of the system to perform one or more of the following tasks: (i) receiving and/or transporting a biological sample from a subject into the system; (ii) adding one or more components, reagents, and/or tools to the biological sample (e.g., one or more components, reagents, and/or tools to perform demethylation and/or PCR and/or sequencing of at least one of the genes listed in Table 1); (iii) performing PCR on the biological sample or a demethylated product thereof; (iv) detecting a PCR product (e.g.
• a PCR product of one or more of the genes listed in Table 1 or a demethylated product thereof (v) sequencing at least one of the genes listed in Table 1 in the biological sample or a demethylated product thereof; (vi) generating a report that indicates the methylation status of one or more genes listed in Table 1 and/or the expression status? of one or more genes listed in Table 1, optionally relative to a control.
• the disclosed systems may include automated systems.
• the disclosed kits may be in any format, including a format suitable for automated systems, such as a cartridge format. Such systems have been described and are well known to the skilled person.
• cartridges for automated systems for detecting methylation analysis of at least one gene are preloaded with reagents and the analysis of promotor methylation is performed in an automated manner by means of a computer-implemented method by an instrument. Once the sample is added to the cartridge, the automated system will run the assay.
• the detection of methylation or promotor methylation may utilize a biological sample comprising nucleic acids or cells comprising nucleic acid to be analyzed according to the disclosed methods. Methods and systems for obtaining nucleic acid from samples have been described and may require isolation and/or purification of the nucleic acid from the sample.
• Detection of promoter methylation may also be determined in a number of alternative ways as the ones exemplified herein.
• This methylation level may be determined by a method selected from the group consisting of PCR, Q-PCR, sequencing, hybridization arrays, restriction enzyme approaches, such as for instance methylation- specific PCR (MSP), quantitative methylation-specific PCR (Q-MSP), Illumina sequencing technology, Oxford Nanopore sequencing technology, Ion TorrentTM sequencing technology, Dreaming technology, High Resolution Melting (HRM) technology, and the like.
• MSP methylation- specific PCR
• Q-MSP quantitative methylation-specific PCR
• Illumina sequencing technology Oxford Nanopore sequencing technology
• Ion TorrentTM sequencing technology Ion TorrentTM sequencing technology
• Dreaming technology High Resolution Melting
• Melt technology uses semi-limiting dilutions and precise melt curve analysis to distinguish and enumerate individual copies of epi-allelic species at single-CpG-site resolution in fractions as low as 0.005%, providing facile and inexpensive ultrasensitive assessment of locus-specific epigenetic heterogeneity directly from liquid biopsies such as stool, urine, and blood samples [6] .
• the disclosed methods may include further steps such as diagnostic steps and treatment steps.
• the disclosed methods comprise performing a colon biopsy on the subject.
• the disclosed methods may comprise administering to the subject a treatment for colorectal cancer, optionally selected from a chemotherapy, a biological agent, a surgery, and/or radiation therapy.
• Example 1 DNA isolation bisulfite conversion and promoter CpG island methylation analyses.
• DNA samples 35ml were centrifuged to remove insoluble parts. DNA was precipitated using ethanol, washed, and dissolved. DNA was isolated using the Qiagen QIAamp Fast DNA stool kit.
• Q-MSP quantitative methylation-specific polymerase chain reaction
• Q-PCR conditions were as follows: The PCR mixture contains 1 x PCR buffer (16.6 mM ammonium sulfate/67 mM Tris, pH 8.8/6.7 mM MgC12/10 mM 2- mercaptoethanol), dNTPs (each at 1.25 mM), primers (300 ng each per reaction), and bisulfite-modified DNA (50 ng) in a final volume of 50 ul. Reactions were hot-started at 95°C for 5 min before the addition of 1.25 units of Taq polymerase (BRL). Amplification was carried out in a thermocycler for 50 cycles (30 sec at 95 °C, 30 sec at the annealing temperature of 64°C, and 30 sec at 72°C), followed by a final 4-min extension at 72°C.
• PCR buffer (16.6 mM ammonium sulfate/67 mM Tris, pH 8.8/6.7 mM MgC12/10 mM 2- mercaptoethanol
• methylated human control DNA for example unmethylated human control DNA, EpiTect Control DNA, Qiagen, Cat. no. 59568
• methylated alleles normal lymphocyte DNA treated in vitro with Sssl methyltransferase [New England Biolabs]
• a no-template DNA control for example unmethylated human control DNA, EpiTect Control DNA, Qiagen, Cat. no. 59568
• methylated alleles normal lymphocyte DNA treated in vitro with Sssl methyltransferase [New England Biolabs]
• no-template DNA control for example unmethylated human control DNA, EpiTect Control DNA, Qiagen, Cat. no. 59568
• methylated alleles normal lymphocyte DNA treated in vitro with Sssl methyltransferase [New England Biolabs]
• Table 9 Probes and primers used for Q PCR.
• Example 2 DNA isolation, bisulfite conversion, and promoter CpG island methylation analyses.
• Q-MSP quantitative methylation-specific polymerase chain reaction
• Q-PCR conditions were as follows: The PCR mixture contains 2x PCR mix (PrimeTime Gene expression Master mix), primers (375 nM each per reaction), probe (250 nM per.reaction), and bisulfite-modified DNA (10 ng) in a final volume of 20 ul. Reactions were performed on a LightCycler 480 (Roche). Polymerase activation for 3 min at 95°C. Amplification was carried out for 45 cycles (15 sec at 95°C and 60 sec at the annealing temperature of 65°C). [00111] Q-PCR reactions were performed with controls for unmethylated alleles
• Double knock out of the HCT-116 human cell line for example Double knock out of the HCT-116 human cell line
• methylated alleles CpG Methylated Human Genomic DNA
• no-template DNA control for example Double knock out of the HCT-116 human cell line
• QIAamp DNA Mini Kit Qiagen, Venlo, The Netherlands.
• Table 14 Sensitivity and specificity of an assay as disclosed herein.
• Example 4 DNA isolation bisulfite conversion and promoter CPG island methylation analyses on blood samples.

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